The main cause of graft rejection is the allo-antigen (Ag)-specific T cell immune response elicited within the recipient. Dendritic cells (DCs) are the professional Ag-presenting cells (APCs) that as "passenger leukocytes" present donor alloAg to naive T cells and trigger graft rejection. Besides their role in immunity and transplant rejection, DCs that have phagocytosed apoptotic vesicles exhibit characteristics of immunoregulatory APCs and seem to be involved in induction of peripheral tolerance in the steady-state. However, apoptotic vesicles are not the only extracellular vesicles that may affect DC biology. DCs can internalize extracellular exosomes, a population of small membrane vesicles (< 100 nm) of endosomal origin, rich in MHC molecules and abundant in the extracellular space. DCs are able to present exosome-derived allopeptides in MHC molecules. There is evidence that exosomes, like apoptotic vesicles, may participate in induction of peripheral tolerance probably through interaction with APCs. We propose to study the DC-exosome interaction and to test the hypothesis that after internalization and processing of donor-derived MHC + exosomes, recipient DCs are able to generate allo-Ag specific T cells with regulatory function. We expect that this interaction will have two important effects: i) recipient DCs will present the donor MHC + exosome-derived allopeptides + self-MHC interfering with the indirect pathway of allorecognition, and ii) due to their low levels of T cell costimulatory molecules, recipient immature DCs expressing donor MHC + exosome-derived allopeptides + self-MHC will instruct recipient alloAg-specific T cells to become apoptotic, anergic, or regulatory T (Treg) cells. Unlike most of the techniques reported in effort to induce transplant tolerance with DCs, our approach targets specifically the indirect pathway of allorecognition in organ rejection. We propose to explore this approach to induce Ag-specific tolerance in heart allograft recipients as an alternative to pharmacological treatment that induces generalized immunosuppression and harmful side effects. The knowledge gained will enhance understanding of how tolerance and rejection are regulated, and will clarify the therapeutic potential of DCs and exosomes for transplantation. We propose the following aims: AIM 1: To investigate the interaction of DCs with exosomes and its impact on DC biology. AIM 2: To study the trafficking of exosomes in vivo and to characterize the T cell stimnlatory capacity of exosome-exposed DCs. AIM 3: To investigate the effect of administration of donor MHC + exosomes or recipient DCs exposed to donor MHC + exosomes on heart allograft survival. To compare the effects of donor-MHC + exosomes with donor-MHC + apoptotic cells.